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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :

C12Q 1/68, C12P 19/34, C07H 21/04,
C07K 13/00

(11) International Publication Number: WO 99/58718

(43) International Publication Date: 18 November 1999 (18.1 1.99)

(21) International Application Number: PCT/ US99/ 10297

(22) International Filing Date: 11 May 1999 ( 1 1 .05.99)

(30) Priority Data:
60/084,944

11 May 1998 (11.05.98)

(71) Applicant (for all designated States except US): QUARK

BIOTECH INC. [US/US]; 1059 Serpentine Lane, Pleasan-
ton, CA 94566 (US).

(72) Inventors; and

(75) Inventors/Applicants (for US only): EINAT, Paz [IU1L];
Neve Nir 1/27, 74042 Nes-Ziona (IL). SKALITER, Rami
[IL/IL1; Habanim 117/10, 74037 Nes-Ziona (IL). MOR,
Orna [IL/IL]; Emek Aialon 12, Ganei Ilan, Kiryat Ono (IL).
LURIA, Sylvie [IL/IL]; Habanim 1 13/17, 70100 Nes-Ziona
(IL). HARRIS, Nicholas [IL/IL]; Hanasi Harishon 14/17,
76302 Rehovot (IL). GROSMAN, Zehava [IL/IL]; Taran
Street 20, 76348 Rehovot (IL).

(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB,
GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG,
KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI,
SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA,
ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ,
UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD,
RU, TJ, TM), European patent (AT, BE, CH, CY, DE, DK,
ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI
patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR,
NE, SN, TD, TG).

Published

With international search report.

(74) Agents: KOHN, Kenneth, I. et al.; Kohn & Associates, Suite
410, 30500 Northwestern Highway, Farmington Hills, MI
48334 (US).

(54) TiUe: METHOD FOR IDENTIFYING GENES

(57) Abstract

A method for identifying genes regulated at the RNA level by cue-induced gene expression. The invention relates to the rapid
isolation of differentially expressed or developmental^ regulated gene sequences through analysis of mRNAs obtained from specific cellular
compartments and comparing the changes in the relative abundance of the mRNA in these compartments as a result of applying a cue
to the tested biological sample. The cellular compartments include polysomal and nonpolysomal fractions, nuclear fractions, cytoplasmic
fractions, and spliceosomal fractions. Genes that are differentially expressed due to regulation on any one or more of a number of levels,
may be characterized. Regulation levels include translational regulation, transcriptional regulation, mRNA stability regulation, and mRNA
transport regulation. A method for identifying gene sequences coding for internal ribosome entry sites is also provided, which includes
inhibiting 5' cap-dependent mRNA translation in a cell, collecting a pool of mRNA from the cells, and differentially analyzing the pool of
mRNA to identify genes with sequences coding for internal ribosome entry sites.

FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.

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WO 99/58718 PCT/US99/10297

METHOD FOR IDENTIFYING GENES
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a conversion of United States Provisional Patent
Application Serial No. 60/084,944, filed May 1 1, 1998, and claims priority
thereon.

BACKGROUND OF THE INVENTION

Technical Field

The present invention relates to a method for identifying genes that
are regulated at the RNA level. More specifically, the present invention relates to
the rapid isolation of differentially expressed or developmentally regulated gene
sequences through analysis of mRNAs obtained from specific cellular
compartments. By comparing changes in the relative abundance of the mRNAs
found in these compartments occurring as a result of application of a cue or
stimulus to the tested biological sample, genes that are differentially expressed can
be characterized.

Background Art

The identification and/or isolation of genes whose expression differs
between two cell or tissue types, or between cells or tissues exposed to stress
conditions, chemical compounds or pathogens, is critical to the understanding of
mechanisms which underlie various physiological conditions, disorders, or
diseases. Regulation of gene expression has been shown to play an important part
in many biological processes including embryogenesis, aging, tissue repair, and
neoplastic transformation. Regulation of gene expression can occur on a number
of levels, including transcriptional regulation, translational regulation, regulation of
mRNA stability, regulation of mRNA transport, regulation by natural antisense
mRNA and regulation by alternative splicing. However, while cases of genes thus
regulated are reported in the literature, the gene discovery approaches followed to
date have only examined changes in the 'steady state' levels of cellular mRNA by
analysis of total cellular RNA.

A number of methods have been developed for the detection and
isolation of genes which are activated or repressed in response to developmental,

WO 99/58718 PCT/US99/10297

physiological, pharmacological, or other cued events. One particular method is
described in United States Patent Number 5,525,471 to Zeng, is subtractive
hybridization. Subtractive hybridization is a particularly useful method for
selectively cloning sequences present in one DNA or RNA population while absent
5 in another, but is less sensitive to more subtle differences. The selective cloning is
accomplished by generating single stranded complementary DNA libraries from
both control cells/tissue (driver cDNA) and cell/tissue during or after a specific
change or response being studied (tester cDNA). The two cDNA libraries are
denatured and hybridized to each other resulting in duplex formation between the

10 driver and tester cDNA strands. In this method, common sequences are removed
and the remaining non-hybridized single-stranded DNA is enriched for sequences
present in the experimental cell/tissue which is related to the particular change or
event being studied. (Davis et al., 1987).

Currently used methodologies to identify mRNAs encoding proteins

15 which are being induced/reduced following a cue or stimulus rely on changes in
steady state mRNA levels via screening of differentially expressed mRNAs. One
such method for the identification of differentially expressed mRNAs is disclosed
in United States Patent Number 5,459,037 to Sutcliffe et al. According to this
method, an mRNA population is isolated, double-stranded cDNAs are prepared

20 from the mRNA population using a mixture of twelve anchor primers, the cDNAs
are cleaved with two restriction endonucleases, and then inserted into a vector in
such an orientation that they are anti-sense with respect to a T3 promotor within
the vector. E. coli are transformed with the cDNA containing vectors, linearized
fragments are generated from the cloned inserts by digestion with at least one
* 25 restriction endonuclease that is different from the first and second restriction

endonucleouseases and a cDNA preparation of the anti-sense cDNA transcripts is
generated by incubating the linearized fragments with a T3 RNA polymerase. The
cDNA population is divided into subpools and the first strand cDNA from each
subpool is transcribed using a thermostable reverse transcriptase and one of sixteen

30 primers. The transcription product of each of the sixteen reaction pools is used as a
template for a polymerase chain reaction (PGR) with a 3'-primer and a 5'-primer

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WO 99/58718 PCT/US99/10297

and the polymerase chain reaction amplified fragments are resolved by
electrophoresis to display bands representing the 3'-ends of the raRNAs present in
the sample. This method is useful for the identification of differentially expressed
mRNAs and the measurement of their relative concentrations. This type of
5 methodology, however, is unable to identify mRNAs whose levels remain constant
but whose translatability is variable or changes, or differences resulting from
changes in mRNA transport from the nucleus to the cytoplasm.

Schena et al. developed a high capacity system to monitor the
expression of many genes in parallel utilizing microarrays. The microairays are

10 prepared by high speed robotic printing of cDNAs on glass providing quantitative
expression measurements of the corresponding genes (Schena et al., 1995).
Differential expression measurements of genes are made by means of
simultaneous, two color fluorescence hybridization. However, this method alone is
of limited sensitivity and is insufficient for the identification of several types of

15 regulation levels, including translationally regulated genes and mRNA transport

regulation. The authors did not examine the use of special mRNA pools that enable
direct assessment of transcriptional activity.

The use of a known inhibitor of hypusine formation, mimosime,
was used to reversibly suppress the hypusine-forming deoxyhypusyl hydroxylase

20 in cells while differentially displaying their polysomal versus non-polysomal

mRNA populations. (Hanauske-Abel et al., 1995) Utilizing this method, several
species of mRNA were discovered which disappear and reappear, respectively, at
polysomes in connection with inhibition and disinhibition of hypusine formation
and which are thought to code for translationally controlled enzymes. This method
* 25 only teaches the use of a known stimulating element (i.e., inducer or repressor) to
identify translationally regulated genes. (This method does not provide a
mechanism for the detection and/or identification of translationally regulated genes
where the stimulating element is unknown). The use of differential display for gene
discovery is very limited in terms of throughput and sensitivity and is prone to

30 many artifices. The subject matter of this paper does not imply the use of

polysomal mRNA pools as sources for probes for DNA chip analysis. This in fact

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WO 99/5871 8 PCT/US99/1 0297

requires special methodological improvements in order to obtain large amounts of
high quality polysomal mRNA.

Generally, the translation of eukaryotic mRNAs is dependent upon
5' cap-mediated ribosome binding. Prior to translation, the ribosome small sub-
unit (40S) binds to the 5'-cap structure on a transcript and then proceeds to scan
along the mRNA molecule to the translation initiation site where the large sub-unit
(60S) forms the complete ribosome initiation site. In most instances, the
translation initiation site is the first AUG codon. This "scanning model" of
translation initiation accommodates most eukaryotic mRNAs. A few notable
exceptions to the "scanning model" are provided by the Picomavirus family.
These viruses produce non-capped transcripts with long (600-1200 nucleotides) 5'-
untranslated regions (UTR) which contain multiple non-translation initiating AUG
codons. Because of the absence of a cap structure, the translational efficiency of
these RNAs is dependent upon the presence of specific sequences within the
untranslated regions (UTR) known as internal ribosome entry sites (IRES).

More recently, IRES containing mRNA transcripts have been
discovered in non-viral systems such as the mRNA encoding for immunoglobulin
heavy chain binding protein, the antenapedia gene in Drosophila, and the mouse
FgI-2 gene. These discoveries have promoted speculation for the role of cap-
independent translation in the developmental regulation of gene expression during
both normal and abnormal processes.

The discovery of the above-mentioned non-viral IRES containing
mRNAs implies that eukaryotic IRES sequences could be more wide spread than
has been previously realized. The difficulty in identifying eukaryotic IRES
sequences resides in the fact that they typically cannot be identified by sequence
homology. [Oh et al., 1993; Mountford et al., 1995; Macejak et al., 1991; Pelletier
et al., 1988; Vagner et al. 1995] It would, therefore, be advantageous to have a
method for identifying IRES containing mRNA in order to identify translationally
controlled genes operating via 5'-cap independent translation in order to ascertain
and assess their association with both normal and abnormal processes.

WO 99/58718 PCT/US99/10297

Prior art methods have only concentrated on very narrow aspects of
gene expression regulation and used methods which have many inherent
limitations. Therefore, it would be desirable to have methods that allow us to
expand the array of gene expression regulation levels and thus enable the isolation
5 of biologically important genes.

SUMMARY OF THE INVENTION

According to the present invention, methods are provided for
identifying genes that may be regulated on a number of possible regulatory levels.

10 Such methods include the steps of exposing cells or tissue to a cue or stimulus such
as mechanical, chemical, toxic, pharmaceutical or other stress, hormones,
physiological disorders or disease; fractionating the cells into compartments such
as polysomes, nuclei, cytoplasm and spliceosomes; extracting the mRNA from
these fractions, and subjecting the mRNA to differential analysis using accepted

15 methodologies, such as gene expression array (GEM).

An example is provided which shows the use of RNA isolation from
nuclei for isolating genes whose steady state levels show only minor changes, but
which show high differential expression when detected by nuclear RNA probe.
Most such genes are regulated at the transcriptional level Another example is

20 provided, of one type of regulation showing the use of polysomes isolated from
cells/tissues to identify genes whose mRNA steady state levels do not change, but
are highly increased in the polysomes after application of a stress cue. Such genes
are regulated strictly on the translation level.

A subgroup of genes regulated on the translational level involves

25 the existence of internal ribosome entry sites. A method is provided for

identification of such genes, which includes inhibiting 5 'cap-dependant mRNA
translation in a cell, collecting a pool of mRNA from the cells, and differentially
analyzing the pool of mRNA to identify genes with sequences coding for internal
ribosome entry sites.

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WO 99/58718 PCT/US99/10297
BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily
appreciated as the same becomes better understood by reference to the following
detailed description when considered in connection with the accompanying
5 drawings wherein:

Figure 1 A is an absorbance profile of a fractionation of cytoplasmic
RNA on a sucrose density gradient wherein the absorbance (at 254nm) is plotted
against the sedimentation rate of the cytoplasmic RNA;

Figure IB is a photograph of purified RNA electrophoresed on an
10 agarous gel and stained with ethidium bromide illustrating the fractionation of
RNA;

Figure 2 is a color representation of DNA chip hybridization results
comparing probes of total RNA to probes derived from polysomal RNA
(translational probes);

15 Figure 3 is a color representation of DNA chip hybridization results

comparing probes of total RNA (Tot) to probes derived from nuclear RNA (STP);

Figures 4A-C are schematic representations of plasmids that contain
the Polio virus 2A genes (A) in plasmid pTK-OP3-WT2A, (B) in the plasmid
miniTK-WT2A, and (C) in a plasmid containing a hygromycin selectable marker;

20 Figure 5 is graph illustrating the induction of

Polio virus 2A protease leading to cell death after induction of the 2A protease;

Figure 6 is a photograph of a gel illustrating the presence of Polio
virus 2A protease expression in transformed HEK-293 cells (293-2A) following
induction with IPTG and the absence of the Polio virus 2A protease in HEK-293

25 (293) parental cells following treatment with IPTG; and

Figure 7 is a photograph of a Western blot illustrating the activity of
the Polio virus 2 A protease in cleaving the p220 protein component of the 40S
ribosomal subunit demonstrating that clones which were induced for Polio virus
2A protease generated cleavage products of the p220 protein.

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WO 99/58718 PCT/US99/10297

DETAILED DESCRIPTION OF THE INVENTION

A method of identifying genes whose expression is regulated at
least in part at the mRNA level by selectively stimulating an unknown target
mRNA with a stress inducing element, the target mRNA being part of a larger
5 sample. The organism may be any organism which provides suitable mRNA. The
mRNA sample is derived from cellular compartments based on expression
regulation and protein localization which are differentially analyzed to identify
genes which are translationally regulated by the stress inducing element. This
method is designed for identifying and cloning genes which are responsive to

10 specific cues. That is, the present method is designed for identifying and cloning
genes which are either up- or down- regulated responsive to a specific pathology,
stress, physiological condition, and so on, and in generally to any factor that can
influence cells or organisms to alter their gene expression.

The method of the present invention provides a novel approach to

15 the identification and cloning of genes that are involved in fundamental cellular
functions and which are regulated at any level in an organism. The basic
underlying theory for this method relies on the knowledge that the regulation of
gene expression can be controlled at different levels (modes) and that each
different regulation levels is manifested by some difference in the distribution of

20 the specific mRNAs in the cell. In genes that are regulated by translation, the

mRNA is stored in the cell in an inactive form and will not be found on polysomes.
Following the appropriate external cue, the mRNA is incorporated into the
polysomes and translated, and the encoded protein quickly appears. By comparing
mRNA populations that are "active" or "non-active" at a given time, genes that are
* 25 regulated by a mechanism referred to as the "shift mechanism" can be identified.

Genes whose main regulatory level is the active transport of mRNA
from the nucleus to the cytoplasm are stored in the nucleus and at the appropriate
cue the mRNA is transported to the cytoplasm. Comparison of mRNA isolated
from the nucleus and cytoplasm before and after the cue can lead to the discovery

30 of genes controlled in this way. The comparison of mRNA derived from the

nucleus also allows direct analysis of the transcription activity of many genes. For

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WO 99/5871 8 PCT/US99/1 0297

most transcriptionally activated genes a basal level of mRNA exists in the cell even
when the basal transcription activity is low. Thus, increased transcription (up to
five-fold) is often obscured when total cellular RNA is used for differential
analysis of gene expression. The use of nuclear RNA allows direct measurement of
5 transcription activity of many genes, since the basal mRNA is found in the
cytoplasm. The result is a major increase in sensitivity for the detection of
differential expression.

In the case of mRNA stability regulation, it is expected that such
mRNA would be similarly transcribed before and after cue administration,

10 resulting in a similar abundance in nuclear mRNA pools. However, if the mRNA is
stabilized following the cue, its abundance in the cytoplasm would become higher.
In the case of mRNA transport regulation, such mRNA is expected to exist at a
high level in the nucleus and a low level in the cytoplasm prior to the cue, which
situation would be reversed after administration of the cue. It is thus easy to

15 differentiate between the two regulatory modes.

The method of the invention includes the identification of genes
regulated at the translational level; genes regulated at the transcription level; genes
regulated by RNA stability; genes regulated by mRNA transport rate between the
nucleus and the cytoplasm; and genes regulated by differential splicing. That is,

20 genes whose expression is at least partly controlled or regulated at the mRNA level
can be identified.

The method will identify genes encoding secreted and membrane
proteins; genes encoding for nuclear proteins; genes encoding for mitochondrial
proteins; and genes encoding for cytoskeletal proteins. In addition, any other gene
' 25 whose expression can be controlled at the mRNA level can be identified by this
method.

As used herein, RNA refers to RNA isolated from cell cultures,
cultured tissues or cells or tissues isolated from organisms which are stimulated,
differentiated, exposed to a chemical compound, are infected with a pathogen or
30 otherwise stimulated. As used herein, translation is defined as the synthesis of
protein on an mRNA template.

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WO 99/58718 PCT/US99/1 0297

As used herein, stimulation of translation, transcription, stability or
transportation of unknown target mRNA or stimulating element, includes
chemically, pathogenically, physically, or otherwise inducing or repressing an
mRNA population from genes which can be derived from native tissues and/or
5 cells under pathological and/or stress conditions. In other words, stimulating the
expression of a gene's mRNA with a stress inducing element or "stressor" can
include the application of an external cue, stimulus, or stimuli which stimulates or
initiates translation of a mRNA stored as untranslated mRNA in the cells from the
sample. The stressor may cause an increase in stability of certain mRNAs, or

10 induce the transport of specific mRNAs from the nucleus to the cytoplasm. The
stressor may also induce gene transcription. In addition to stimulating translation
of mRNA from genes in native cells/tissues, stimulation can include induction
and/or repression of genes under pathological and/or stress conditions. The present
method utilizes a stimulus or stressor to identify unknown target genes which are

15 regulated at the various possible levels by the stress inducing element or stressor.

The method of the present invention synergistically integrates two
types of previously known methodologies which were otherwise used separately.
The first method is the division of cellular mRNA into separate pools of mRNA
derived from polysomes, nucleus, cytoplasm or spliceosomes. The second

20 methodology involves the simultaneous comparison of the relative abundance of
the mRNA species found in the separate pools by a method of differential analysis
such as differential display, representational difference analysis (RDA), gene
expression microarray (GEM), suppressive subtraction hybridization (SSH)
(Diatchenko et al, 1996), and oligonucleotide chip techniques such as the chip
' 25 technology exemplified by United States Patent No. 5,545,53 1 to Rava et al.

assigned to Afiymax Technologies N.V. and direct sequencing exemplified by WO
96/17957 patent application to Hyseq, Inc.

Briefly, subtractive hybridization is defined as subtraction of
mRNA by hybridization in solution. RNAs that are common to the two pools form

30 a duplex that can be removed, enriching for RNAs that are unique or more

abundant in one pool. Differential Display is defined as reverse transcription of

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WO 99/58718 PCT/US99/10297

mRNA into cDNA and PCR amplification with degenerated primers. Comparison
of the amounts amplification products (by electrophoresis) from two pools indicate
transcript abundance. RDA, GEM, SSH, SAGE are described herein above.

The specific cells/tissues which are to be analyzed in order to
identify translationally regulated genes, can include any suitable cells and/or
tissues. Any cell type or tissue can be used, whether an established cell line or
culture or whether directly isolated from an exposed organism.

The cells/tissues to be analyzed under the present method are
selectively stimulated or "stressed" utilizing a physiological, chemical,
environmental and/or pathological stress inducing element or stressor, in order to
stimulate the translation of mRNA within the sample tissue and identify genes
whose expression is regulated at least in part at the mRNA level. Stimulation can
cause up or down regulation. Following stimulation, RNA is isolated or extracted
from the cells/tissues. The isolation of the RNA can be performed utilizing
techniques which are well known to those skilled in the art and are described, for
example, in "Molecular Cloning; A Laboratory Manual" (Cold Springs Harbor
Laboratory Press, Cold Spring Harbor, New York, 1989). Other methods for the
isolation and extraction of RNA from cells/tissue can be used and will be known to
those of ordinary skill in the art. (Mach et al., 1986, Jefferies et al., 1994).
However, may variations of these methodologies have been published. The
methods described herein were carefully selected after many trials.

The mRNAs which are actively engaged in translation and those
which remain untranslated can be separated utilizing a procedure such as
fractionation on a sucrose density gradient, high performance gel filtration
chromatography, or polyacrylamide gel matrix separation (Ogishima et al., 1984,
Menaker et al., 1974, Hirama et al., 1986, Mechler, 1987, and Bharucha and
Murthy, 1992), since mRNAs that are being translated are loaded with ribosomes
and, therefore, will migrate differently on a density gradient than ribosome-free
untranslated mRNAs. By comparing mRNA populations that are active or non-
active in translation at a given time, genes that are regulated by the "shift
mechanism" can be identified.

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WO 99/58718 PCT/US99/10297

Polysomal fractionation and specific analysis can be facilitated by
treatment of target cell/tissue with drugs that will specifically inhibit or modulate
transcription or translation. Examples of such drugs are actinomycin D and
cyclohexamide, respectively.
5 The fractionation can be completed to create polysomal

subdivisions. The subdivisions can be made to discriminate between total
polyribosomes or membrane bound ribosomes by methods known in the art
(Mechler, 1987). Further, the mRNA sample can additionally be fractionated into
one or more of at least the following subsegments or fractions: cytoplasmatic,

10 nuclear, polyribosomal, sub polyribosomal, microsomal or rough endoplasmic

reticulum, mitochondrial and splicesome associated mRNA by methods known in
the art (see also Table 1).

More specifically, nuclear fractions can be obtained using the
method set forth in the article entitled Abundant Nuclear Ribonucleoprotein Form

15 of CAD RNA (Sperling, 1984) as set forth in the Experimental section, thus

allowing nuclear RNA to be utilized for a method of identifying genes which are
regulated or responsive to stress conditions. Further, antisense RNA can be utilized
as a method for identifying genes which are responsive to specific pathology or
stress conditions. Antisense RNA can be isolated using the methods described by

20 Dimitrijevic, whose abstract details the methods utilized for obtaining and isolating
antisense RNA from a sample. Additionally, microsomal fractions may be
obtained using the methods of the present invention as set forth in the
Experimental Section which are modifications of the methods disclosed by Walter
andBlobelinl983.

* 25 Following isolation and division of the total mRNA population into

separate expression regulation and protein localization pools of mRNA, the relative
abundance of the many mRNA species found in these pools are simultaneously
compared using a differential analysis technique such as differential display,
oligonucleotide chips, representational difference analysis (RDA), GEM-Gene
30 Expression Microarrays (Schena et al., 1995, Aiello et al., 1994, Shen et al., 1995,
Bauer et al., 1993, Liang and Pardee, 1992, Liang and Pardee, 1995, Liang et al.,

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WO 99/58718 PCT/US99/ 10297

1993, Braun et al., 1995, Hubank and Schatz, 1994) and suppressive subtraction
hybridization (SSH). The RNA isolated from the fractions can be further purified
into mRNA without the ribosomal RNA by poly A selection. It should be noted
that multiple pools can be analyzed utilizing this method. That is, different cell
aliquots subjected to different stressors can be compared with each other as well as
with the reference sample.

Labeled nucleic acid probes (in a cDNA JPCR product or rRNA
transcribed from the cDNA) made from RNA derived from polysomal, non-
polysomal, mRNPs, nuclear, cytoplasmic, or spliceosome fractions can be used as
probes, to identify clones of cDNA, genomic clones, and mRNA species that are
fixed onto a solid matrix-like microarrays such as (GEM), that shown in United
States Patent Number 5,545,531 to Rava et al. and W096/17957 to Hyseq, Inc.,
and membranes of any kind where clones can be either blotted after electrophoresis
or directly loaded (dot blot) onto the membrane. The label can be radioactive,
fluorescent, or incorporating a modified base such as digoxigenin and biotin.

Comparison between the fractions derived from the polysomal or
polyribosomal fraction or other fractions to the total unfractionated material is
essential to discriminate between differentials in expression levels that are the
result of transcription modulation from those that result from modulation of
translation per se. The polysomal fractions or groups can include membrane bound
polysomes, loose or tight polysomes, or free unbound polysome groups.

The importance of utilizing the polysomal sub-population in order
to identify differentially (translationally) expressed genes is shown in Example 1
where a number of genes were not detected as translationally expressed under heat
shock inducement when total mRNA was used as the detection probe but, however,
when polysomal mRNA was used as a probe, a number of genes were identified as
differentially expressed. As shown in Example 1, a number of genes under heat
shock inducement with total mRNA derived probe were detected when probed with
polysomal mRNA fractions. Heat shock, being a model for acute diseases such as
ischemic diseases, reveal the importance of the polysomal probe. Cells store
critical mRNAs in an inactive form so that in an acute situation they can be quickly

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WO 99/5871 8 PCT/US99/1 0297

loaded onto polysomes (without the need to wait for their production by
transcription) and translated to produce the proteins the cells require for their
survival under stress.

The present method for identifying translationally regulated genes is
5 not limited by the source of the mRNA pools. Therefore, the present method can
be utilized to clone genes from native cells/tissue under pathological and/or stress
conditions that are regulated by the "shift mechanism," as well as genes that are
induced/repressed under pathological and/or stress conditions. Pathologies can
include disease states including those diseases caused by pathogens and trauma.
10 Stress conditions can also include disease states, physical and psychological

trauma, and environmental stresses. Following analysis by the selected method of
differential analysis, the genes which have been identified as being regulated by
translation can be cloned by any suitable cloning methodologies known to those
skilled in the art. (Lisitsyn and Wigler, 1993).
15 Differential comparisons can be made of all possible permutations

of polysomal vs. non-polysomal RNA where the definition of the fraction type is
done, for example, by absorbance profile at 254nm, density of the sucrose gradient
as shown in Figure 1 A (or another size standard if high pressure liquid
chromatography or gel systems are used) and types of RNA that are stained with
20 ethidium bromide after electrophoresis of the fractions on agarous gels are

completed, as shown in Figure IB. In Figure 1 A, the polysomal fractions are those
that have mRNA with more than two ribosomes loaded. The materials and
methods for this comparison are set forth below in the experimental section.

Differential comparisons can also include polysomal vs. non-
25 polysomal fractions in each condition. By "condition" it is meant that cells from
the same source, such as a cell line, a primary cell, or a tissue that undergoes
different treatment or has been modified to have different features or to express
different sets of genes. For example, this can be accomplished by differentiation,
transformation, application of the stress such as oxygen deprivation, chemical
30 treatment, or radiation. Permutations can include, for example:

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WO 99/58718 PCT/US99/10297

1. polysomal fractions between conditions individually (migrating
in the same density) or in a pool;

2. non-polysomal fractions between conditions individually
(migrating in the same density) or in a pool;

5 3. non-polysomal to polysomal between conditions and within each

condition individually (migrating in the same density) or in a pool; and

4. each of the fractions being polysomal and non-polysomal
individually (migrating in the same density) or in a pool that can be compared to
total RNA that is unfiactionated.

10 The method described above for the identification of genes

regulated on the translational level has a number of applications. A particular
application for this method is its use for the detection of changes in the pattern of
mRNA expression in cells/tissue associated with any physiological or pathological
change. By comparing the translated versus untranslated mRNAs, the effect of the

15 physiological or pathological cue or stress on the change of the pattern of mRNA
expression in the cell/tissue can be observed and/or detected. This method can be
used to study the effects of a number of cues, stimuli, or stressors to ascertain their
effect or contribution to various physiological and pathological activities of the
cell/tissue. In particular, the present method can be used to analyze the results of

20 the administrations of pharmaceuticals (drugs) or other chemicals to an individual
by comparing the mRNA pattern of a tissue before and after the administration of
the drug or chemical. This analysis allows for the identification of drugs,
chemicals, or other stimuli which affect cells/tissue at the level of translational
regulation. Utilizing this method, it is possible to ascertain if particular mRNA
• 25 species are involved in particular physiological or disease states and, in particular,
to ascertain the specific cells/tissue wherein the external stimulus, i.e., a drug,
affects a gene which is regulated at the translational level.

The identification of a subgroup of genes regulated on the
translational level involved a method for identifying gene sequences coding for

30 internal ribosome entry sites (IRES), including the general steps of inhibiting

5'cap-dependant mRNA translation in a cell, collecting a pool of mRNA from the

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WO 99/58718 PCT/US99/10297

cells, and differentially analyzing the pool of mRNA to identify genes with
sequences coding for internal ribosome entry sites.

As described above, it is known that an exception to the standard 5'-
cap dependent translation initiation exists. Sequences exist within untranslated
regions (UTRs) of RNAs which can include the presence of specific sequences
known as internal ribosome entry sites (IRES). (Ehrenfeld, 1996) These internal
ribosome entry sites have been shown to support translation initiation for several
prokaryotic and eukaiyotic systems as set forth above. However, in order to
identify translationally controlled genes via 5'-cap independent translation
mechanisms and their association with both normal and abnormal processes, it is
necessary to inhibit 5'-cap initiated translation so that 5'-cap independent mRNA
translation can be selected for . This inhibition is necessary since IRES sequences
are difficult, if not impossible, to identify by sequence homology.

In order to inhibit 5'-cap dependent translation and thereby select
for the presence of 5'-cap independent translation, cells or tissues which are to be
analyzed for the presence of internal ribosome entry sites must be treated in some
manner to prevent or discourage the 5'-cap translation initiation mechanism. The
mechanism(s) of standard scanning-type translation initiation should be
substantially, if not totally, turned off or shut down to, in essence, shift the
translation equilibrium in favor of IRES initiated translation. That is, recognition
of the 5'-cap structure is inhibited by disrupting the normal mechanism for 5'-cap
mediated initiation. The mechanism for inhibiting the 5'-cap translation can
include any known means or mechanisms for preventing the initiation of 5'-cap
mediated translation. One such mechanism for inhibiting 5'-cap mediated
translation is the expression of Polio virus 2 A protease into a cell, cell system, or
tissue to be analyzed for the presence of IRES sequences. The use of the Polio
virus 2A protease inhibits 5'-cap-dependent mRNA translation by inactivating the
cellular 5'-cap-dependent translation machinery. This enables the identification of
cellular IRES containing genes which may be translationally controlled and play a
critical role in the immediate response of the cell following the application of a
stress inducing element/stressor such as heat shock, hypoxia, or other stress

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WO 99/58718 PCT/US99/1 0297

inducing elements as set forth above, prior to gene activation. The Polio virus 2A
protease prevents 5'-cap-mediated translation by cleaving the large sub-unit of elF-
4y (p220) of eukaryotic translation initiation factor 4 (eIF-4) which is involved in
the recognition of the mRNA 5' -cap.

In order to inhibit the 5'-cap-mediated translation, the Polio virus
2A protease must be incorporated into the cell or cells being analyzed for the
presence of gene sequences coding for internal ribosome entry sites and/or for
identifying translationally regulated genes. One such method for incorporating the
Polio virus 2A protease into a cell involves the transformation of a target cell with
an expression vector containing the gene which codes for the Polio virus 2A
protease. Because the Polio virus 2A protease is deleterious to living cells when it
is constitutively expressed, the expression vector containing the Polio virus 2A
protease gene is coupled with a bacterial Lad inducible system wherein a LacI
repressor is constituitively expressed under a CMV promoter. The Polio virus 2 A
protease may be expressed under a number of suitable promoters including the
RSV, the TK, or the mini-TK promoter coupled at their 3' end to the LacI repressor
binding sites. By transforming the target cells with an expression vector
containing the LacI repressor and the Polio virus 2A expression vector, the
expression of the Polio virus 2A protease can be induced upon treatment of the
cells with isopropyl-P-D-thiogalatopyranoside (IPTG). Treatment of the target
cells with IPTG relieves the binding of the LacI repressor molecules bound at the
repressor binding sites thus enabling transcription of the Polio virus 2A protease.
By coupling the expression of the Polio virus 2A protease to an inducible system,
such as the LacI system, this mechanism allows for the establishment of control of
the expression of the gene coding for the Polio virus 2A protease.

Following induction of the expression of the Polio virus 2 A
protease in the target cells, UNA, presumably containing internal ribosome entry
sites, can be collected and analyzed utilizing the methods described above to
identify genes whose translation is up-regulated by the effects of the Polio virus 2A
protease.

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EXPERIMENTAL

DIFFERENTIAL TRANSLATION

MATERIALS AND METHODS
General Scheme

a. Total mRNA organic extraction of all RNA from the source tissue or cell,
(additional selection for polyA+ mRNA can be included).

b. Nuclear RNA-lysis of cells (from a tissue or a cell line) by homogenization in
hypotonic buffer. Collection of nuclei by centrifiigation and organic extraction of
the RNA.

c. Cytoplasmic RNA - Organic extraction of the RNA from the supernatant from b
above.

d. Polyribosomal/subpolyribosomal fractionation. Lysis of cells by
homogenization hypotonic buffer, removal of nuclei and fractionation of
polyribosome on linear sucrose gradients and organic extraction of the RNA from
each fraction of the gradient

e. Secreted and membrane encoding transcripts.

1. Isolation of RER on Percol gradients (after homogenization of cells).

2. Preparation of microsomes containing the RER

3. Isolation of membrane-bound polyribosomes by successive treatment of cells
with detergents.

f. Nuclear proteins. Isolation of cytoskeletal associated polyribosomes by treating
cells lyzates with different detergents.

g. Mitochondrial genes. Isolation of mitochondria on Percoll gradients.

h. Alternative splicing. Separation of nuclei and isolation of splicsosome (proteins
and RNA complex) on linear sucrose gradients.

Preparation of cell extracts

Cells were centrifiiged. The pellet was washed with PBS and recentrifuged. The
cells were resuspended in 4x of one packed cell volume (PCV) with hypotonic
lysis buffer (HLB: 20mM TrisHCL pH=7.4; lOmM NaCl; 3mM MgCl 2 ). The
cells were incubated five minutes on ice. lxPCV of HLB containing 1 .2% Triton

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WO 99/58718 PCT7US99/10297
X-100 and 0.2M sucrose was added. The cells were homogenized with a Dounce
homogenizer (five strokes with B pestle). The cell lysate was centrifuged at 2300g
for ten minutes at 4°C. The supernatant was transferred to a new tube. HLB
containing lOmg/ml heparin was added to a final concentration of lmg/ml heparin.
5 NaCl was added to a final concentration of 0. 1 5M. The supernatant was frozen at
-70°C after quick freezing in liquid N 2 or used immediately.

Sucrose gradient fractionation

A linear sucrose gradient from 0.5M to 1.5M sucrose in HLB was prepared,
10 Polyallomer tubes (1 4X89mm) were used. 0.5 to 1 .0ml of cell extract was loaded
on the gradient. The cells were centrifuged at 36,000 RPM for 1 10 minutes at 4°C.
An ISCO Density Fractionator was used to collect the fractions and record the
absorbance profile.

15 RNA purification

SDS was added to 0.5% and Proteinase K to 0. lmg/ml and incubated at 37°C for
30 minutes. Extract with an equal volume of phenol+chloroform (1:1). The
aqueous phase was extracted with one volume of chloroform and the RNA was
precipitated by adding Na- Acetate to 0.3M and 2.5 volumes of ethanol and

20 incubating at -20°C overnight. Centrifuged ten minutes, the supernatant was
aspirated and the RNA pellet was dissolved in sterile, diethylpyrocarbonate
(hereinafter referred to as "DEPC") DEPC-treated water.

Preparation of Microsomes
- 25 When possible fresh tissues and cells are used, without freezing. Tissues were
powdered in liquid nitrogen with mortar and pestle and then homogenized using
4ml of buffer A/1 gr tissue (Buffer A is 250mM sucrose, 50mM TEA, 50mM
KOAc pH7.5, 6niM MgtOacX, ImM EDTA, ImM DTT, 0.5mM PMSF. PMSF
was made in ethanol before making the buffer and added in drops to buffer while
30 being stirred. This was stirred for 1 5 minutes and then DTT was added). Fresh
organs were washed in Buffer A a few times, and then cut into pieces and

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homogenized Approximately 5ml buffer A/5xl 0 8 cells were added and
homogenized. This was then homogenized on ice for 5-10 times, or as needed with
the individual tissue. The mixture was transferred to 50ml tubes, then centrifuged
for 10 minutes, at 4°C in a swinging bucket rotor machine. Next, the supernatant
was transferred, avoiding the pellet as much as possible, to a Sorvall tube, the
pellet was washed again with 1ml buffer and centrifuge as before. The two pellets
were combined, thus establishing the nuclear fraction. The combination was
dissolved and treated the pellet with Tri-reagent (usually 2ml of Tri-reagent when
sample is from cells) to extract the nuclear RNA. The combined 1st and 2nd
supernatants were centrifuged for 10 minutes at lOOOOg at 4°C. Again, the
supernatant was transferred to a tube and kept on ice. The pellet was washed again
with 1ml buffer and centrifuged for 10 minutes at lOOOOg and the two pellets were
combined as before, thus establishing the Mitochondrial pellet. Again, the pellet
was treated with Tri-reagent (usually 1ml with cells) and the Mitochondrial RNA
was extracted. Next, cold ultracentrifuge tubes were prepared containing a sucrose
cushion made of: buffer A + L3M sucrose. The volume of the cushion was
approximately 1/3 of the supernatant. The supernatant was loaded on the cushion
in a 1 :3 ratio of cushion to supernatant. A pair of tubes was weighed for balancing,
a 20-30mg difference is allowable. The tubes were centrifuged 2.5 hours at
140,000g, 4°C with a Ti60.2 rotor (45,000 rpm). When two phases of supernatant
were visible, then the red phase only was transferred (if possible), as the
cytoplasmic fraction, to a sorvall tube. The clear supernatant was aspirated. When
not possible to separate or phase distinction not visible, all the supernatant was
taken as cytoplasmic fraction and dilute sucrose with TE (lOmM Tris-HCl pH 8.0,
ImM EDTA). In the pellet were the microsomes which were visible and were
clear or yellowish. For the RNA extraction, the cytoplasmic fraction was treated
with 1% SDS, O.lmg/ml proteinase K, for 30 minutes, at 37°C. After this, freezing
at -80°C was possible. The RNA was extracted with a phenolxhloroform
combination and precipitate with 0.3M Na-acetate, 1^1 glycogen, and equal
volume of isopropanol O'N precipitation was possible and can be accomplished at
30 minutes on ice. The extract was spun at 1 OOOOg, for 20 minutes, then the RNA

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WO 99/5871 8 PCT/US99/1 0297

pellet was washed with 70% ethanol. The pellet was dried and then dissolved in
H 2 0. The microsomes were then dissolved with 0.1M NaCl/1% SDS solution (1ml
is usually sufficient for a small pellet) and extracted with a phenolrchloroform
combination (no proteinase K treatment). Then the precipitation of the RNA was
done in the same way as for the cytoplasmic fraction but without the requirement
of adding salt.

Preparation of Nuclear and Cytoplasmic RNA

Subconfluent plates were washed with 125 mM KC1-30 mM Tris-hydrochloride
(pH 7.5)-5 mM magnesium acetate- 1 mM 2-mercaptoethanol-2 mM ribonucleoside
vanadyl complex (2)-0.15 mM spermine-0.05 mM spermidine at 4°C, and cells
scraped from the plates were washed twice with the same buffer. Approximately
10 8 cells were allowed to swell for 10 minutes in 2.5 ml of swelling buffer (same as
wash buffer except the KCI concentration was 10 mM) lysed with 20 strokes of a
Dounce homogenizer (B pestle), overlaid on an equal volume of swelling buffer
containing 25% glycerol, and centrifuged for 5 min. at 400 x g and 4°C. The upper
layer of the supernatant, which contained 90% of the CAD sequences released by
lysis, was designated the cytoplasmic fraction. The nuclear pellet was washed
once with 2 ml of swelling buffer-25% glycerol-0.5% Triton X-100 and once with
2 ml of swelling buffer.

Nuclear RNP. Nuclei from 10 8 cells, prepared as described above,
were suspended in 1 ml of 10 mM Tris-hydrochloride (pH 8.0)-100 mM NaCl-2
mM MgCl 2 -l mM 2-mercapthoethanol-0. 1 5 mM spermine-0.05 mM spermidine-
10 mM ribonucleoside vanadyl complex (2)-100 U of placental RNase inhibitor
(Amersham Corp.) per ml and sonicated at the maximum power setting of a Konres
micro-ultrasonic cell disrupter for 20 g at 4°C. Bacterial tRNA (2 mg) was added,
to adsorb basic proteins (9), and the mixture was centrifuged for 1 minute
(Eppendorf microcentrifuge). The supernatant was applied to a 15 to 45% sucrose
gradient in mM Tris-hydrochloride- 1 00 mM NaCl-2 mM MgCl 2 -2 mM
ribonucleoside vanadyl complex and centrifuged in a Beckman SW41 rotor for 90
minutes at 40,000 rpm and 4°C. RNA was recovered from gradient fractions by the

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WO 99/58718 PCT/US99/ 10297

addition of sodium dodecyl sulfate to 0.5%, treatment with proteinase K (200
jig/ml) for 2 hours at 37°C, extraction with phenol, and precipitation with ethanol

Preparation of Antisense RNA

Total cellular RNA is extracted. Part of the RNA pool is immobilized on a
membrane, another part converted into cDNA after ligation of
oligodeoxynucletides to the 3 '-ends. The use of biotinylated, complementary
oligos for cDNA synthesis allows immobilization of a "minus" strand to
streptavidin-coated magnetic beads. A second set of oligos is ligated to the cDNA
at the previous 5'-end of the RNA. Plus strands are eluted from the bound strands
and hybridized to the membrane-bound RNA. Since the cDNA strand used has the
same polarity of the RNAs, only cDNA sequences that can bind to complementary
RNAs should be retained. PCR amplification and subsequent cloning of PCR-
fragments is followed by sequence analysis. To test whether cloned sequences are
correctly identified, probes are generated in sense and antisense direction. Positive
clones will be structurally and functionally characterized. In order to work out this
method, we started using a bacterial strain (Escherichia coli), containing plasmid
Rl that regulates its copy number by antisense RNA. Previous work has identified
both antisense (CopA) and target RNA (CopT) of Rl intracellularly. This
procedure, if feasible, will then be used to screen for antisense RNA systems in
other organisms.

DIFFERENTIAL ANALYSIS
Differential display:

Reverse transcription: 2jxg of RNA were annealed with Ipmol of oligo dT primer
(dT) I8 in a volume of 6.5^1 by heating to 70°C for five minutes and cooling on ice.
2^1 reaction buffer (x5), l^il of lOmM dNTP mix, and 0.5^1 of Superscript II
reverse transcriptase (GibcoBRL) was added. The reaction was carried out for one
hour at 42°C. The reaction was stopped by adding 70|il TE (lOmM Tris pH=8;
O.lmMEDTA). Oligonucleotides used for Differential display: The
oligonucleotides were essentially those described in the Delta RNA Fingerprinting

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WO 99/58718 PCT7US99/10297

kit (Clonetech Labs. Inc.). There were 9 "T" oligonucleotides of the structure: 5'
CATTATGCTGAGTGATATCTTTTTTTTTXY 3' (SEQ ID No: 1). The 10 "P"
oligonucleotides were of the structure: 3' ATTAACCCTCACTAAA
"TGCTGGGGA" 3' (SEQ ID No: 1 1) where the 9 or 10 nucleotides between the
5 parenthesis represent an arbitrary sequence and there are 1 0 different sequences
(SEQ ID Nos. 12-21), one for each T oligo.

Amplification reactions : each reaction is done in 20^1 and contains 50^iM dNTP
mix, \\iM from each primer, lx polymerase buffer, 1 unit expand Polymerase
10 (Beohringer Mannheim), 2^Ci [<x- 32 P]dATP and cDNA template. Cycling
conditions were: three minutes at 95°C, then

three cycles of two minutes at 94°C, five minutes at 40°C, five minutes at 68°C.
This was followed by 27 cycles of one minute at 94°C, two minutes at 60°C, two
minutes at 68°C. Reactions were terminated by a
15 seven minute incubation at 68°C and addition of 20^1 sequencing stop solution
(95% fonnamide, lQmM NaOH, 0.025% bromophenol blue, 0.025% xylene
cyanol).

Gel analysis : 3-4jil were loaded onto a 5% sequencing polyacrylamide gel and
20 samples were electrophoresed at 2000 volts/40 milliamperes until the slow dye
(xylene cyanol) was about 2 cm from the bottom. The gel was transferred to a
filter paper, dried under vacuum and exposed to x-ray film.

Recovery of differential bands : bands showing any a differential between the
* 25 various pools were excised out of the dried gel and placed in a microcentrifuge
tube. 50^1 of sterile H 2 0 were added and the tubes heated to 100°c for five
minutes, l^il was added to a 49|il PCR reaction using the same primers used for
the differential display and the samples were amplified for 30 cycles of: one
minute at 94°C, one minute at 60°C and one minute at 68°C. lOfxl was analyzed
30 on agarous gel to visualize and confirm successful amplification.

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WO 99/58718 PCT/US99/10297
REPRESENTATIONAL DIFFERENCE ANALYSIS
Reverse transcription : as above but with 2\xg polyA+ selected mRNA.
Preparation of double stranded cDNA: cDNA from previous step was treated with
alkali to remove the mRNA, precipitated and dissolved in 20)il H 2 0. 5\x\ buffer,
2|il lOmM dATP, H 2 0 to 48|il and 2\i\ terminal deoxynucleotide transferase
(TdT) were added. The reaction was incubated 2-4 hours at 37°C. 5^1 oligo dT
(1 \ig/\i\) was added and incubated at 60°C for 5 minutes. 5jlx1 200 raM DTT, 1 0 |il
lOx section buffer (lOOmM Mg Cl 2 , 900 mM Hepes, pH 6.6) 16 |xl dNTPs (1
raM), and 16 U of Klenow were added and the mixture was incubated overnight at
room temperature to generate ds cDNA. 100^1 TE was added and extracted with
phenol/chloroform. The DNA was precipitated and dissolved in 50fal H 2 0.

Generation of representations : cDNA with DpnII was digested by adding 3\i\

DpnII reaction buffer 20 V and DpnII to 25|il cDNA and incubated five hours at

37°C. 50^1 TE was added and extracted with phenol/chloroform. cDNA was

precipitated and dissolved to a concentration of 10ng/)il .

The following oligonucleotides are used in this procedure:

R-Bgl-12 5' GATCTGCGGTGA 3' (SEQ ID No: 22)

R-Bgl-24 5' AGCACTCTCCAGCCTCTCACCGCA 3' (SEQ ID No:23)

J-Bgl-12 5' GATCTGTTCATG 3' (SEQ ID No: 24)

J-Bgl-24 5' ACCGACGTCGACTATCCATGAACA 3' (SEQ ID No:25)

N-Bgl-12 5' GATCTTCCCTCG 3' (SEQ ID No:26)

N-Bgl-24 5' AGGCAACTGTGCTATCCGAGGGAA 3' (SEQ IDNo:27)

R-Bgl-12 and R-Bgl-24 oligos were ligated to Tester and Driver: 1 .2|ig DpnII
digested cDNA. 4\xl from each oligo and 5\il ligation buffer X10 and annealed at
60°C for ten minutes. 2^1 ligase was added and incubated overnight at 16°C. The
ligation mixture was diluted by adding 140jil TE. Amplification was carried out
in a volume of 200|al using R-Bgl-24 primer and 2^1 ligation product and
repeated in twenty tubes for each sample. Before adding Taq DNA polymerase,
the tubes were heated to 72°C for three minutes. PGR conditions were as follows:

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WO 99/58718 PCI7US99/10297

five minutes at 72°C, twenty cycles of one minute at 95°C and three minutes at
72°C, followed by ten minutes at 72°C.

Every four reactions were combined, extracted with phenol/chloroform and
precipitated. Amplified DNA was dissolved to a concentration of 0.5|ag/jxl and all
5 samples were pooled.

Subtraction : Tester DNA (20^ig) was digested with DpnII as above and separated
on a 1.2% agarous gel. The DNA was extracted from the gel and 2\ig was ligated
to J-Bgl-12 and J-Bgl24 oligos as described above for the R-oligos. The ligated

10 Tester DNA was diluted to 10ng/^l with TE. Driver DNA was digested with
DpnII and repurified to a final concentration of 0.5^ig/^ 1 . Mix 40^g of Driver
DNA with 0.4ng of Tester DNA. Extraction was carried out with
phenol/chloroform and precipitated using two washes with 70% ethanol,
resuspended DNA in 4jal of 30mM EPPS pH=8.0, 3mM EDTA and overlayed

15 with 35^1 mineral oil. Denatured at 98°C for five minutes, cool to 67°C and 1 p.1
of 5M NaCl was added to the DNA. Incubated at 67°C for twenty hours. Diluted
DNA by adding 400jxl TE.

Amplification : Amplification of subtracted DNA in a final volume of 200^x1 as
20 follows: Buffer, nucleotides and 20nl of the diluted DNA were added, heated to
72°C, and Taq DNA polymerase was added. Incubated at 72°C for five minutes
and added J-Bgl-24 oligo. Ten cycles of one minute at 95°C, three minutes at
70°C were performed. Incubated ten minutes at 72°C. The amplification was
repeated in four separate tubes. The amplified DNA was extracted with
* 25 phenol/chloroform, precipitated and all four tubes were combined in 40nl
0.2XTE, Digested with Mung Bean Nuclease as follows: To 20nl DNA 4\i\
buffer, 14jxl H 2 0 and 2^1 Mung Bean Nuclease (10 units/jil) was added.
Incubated at 30°C for thirty-five minutes + First Differential Product (DPI).

30 Repeat subtraction hybridization and PCR amplification at driver : differential ratio
of 1 :400 (DPII) and 1 :40,000 (DPIII) using N-Bgl oligonucleotides and J-Bgl

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WO 99/58718 PCTAJS99/10297
oligonucleotides, respectively. Differential products were cloned into a Bluescript
vector at the BAM HI site for analysis of the individual clones.

EXAMPLES

EXAMPLE 1

Analysis of Genes Regulated at a Translational Level in a Representative Heat
Shock GEM Differential Expression System
Materials and Methods

The experimental cells were grown under both normal temperature
(37°C) and heat shock temperature (43°C) for four hours. The cells were then
harvested and cytoplasmic extracts were obtained, polysomes were fractionated
and RNA extracted therefrom. From parallel cultures of cells, total cellular RNA
was extractedThen, the extracted RNA was analyzed utilizing GEM technology as
disclosed above.

Figure 2 and Tables 2 and 3 demonstrate the utility of utilizing
polysomal probes versus total mRNA probes in differential expression analysis to
identify genes which are differentially expressed in response to a stimulus such as
heat shock. These Tables illustrate that fibronectin, pyruvate kinase, protein
disulfide isomerese, poly(ADPribose) polymerase, thymopoietin, 90Kd heat shock
protein, acylamino acid-releasing enzyme, p-spectrin, and pyruvate kinase were all
identified as being differentially expressed utilizing a polysomal probe whereas,
with the exception of fibronectin, the other proteins were not identified as being
differentially expressed when a total mRNA probe was utilized. This example
demonstrates the utility of the present invention for identifying translationally or
differentially regulated genes which are regulated by a stress inducing element.
Additionally, in Table 2, the results of heat shock differential gene expression
analysis with both polysomal probes and total mRNA probes is provided. Table 2
illustrates that a number of differentially expressed genes were identified using a
polysomal probe whereas when a total mRNA probe was used, these genes were
not necessarily identified as being differentially expressed. Table 3 statistically
illustrates the number of differentially expressed genes identified utilizing either

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total mRNA or polysomal mRNA as a probe. Table 3 clearly illustrates that
polysomal mRNA probes yielded between two and greater than ten fold increases
in the number of differentially expressed genes versus total mRNA probes.

5 EXAMPLE 2

Analysis of Genes at a Transcriptional Level vising Nuclear mRNA Probes

The experimental cells were grown alternatively under normal
conditions, for 4 hours under hypoxia (<1% oxygen) and for 16 hours under
hypoxia. The cells were harvested and RNA was extracted either from nuclei that
10 were prepared from the cells (nuclear RNA) or from extracts of unfractionated cells
(total cellular RNA).

Figure 3 demonstrates how the probes prepared from the nuclear
RNA (STP) give a higher differential expression than the total cellular RNA probe
(Tot). The control genes encoding VEGF (vascular endothelial growth factor),
15 Glutl (glucose transporter 1) and glycogen synthase are known to be induced by
the hypoxia stress. The level of induction observed in the nuclear probe is much
higher than that seen in the total probe and much closer to the actual know level of
induction. The three new genes RTP 241, RTP 262 and RTP 779 show marked
induction by hypoxia. Again, the induction level seen with the nuclear probe is
20 much higher, up to five-fold higher, as seen for RTP779. When the induction of
these genes was analyzed by the Northern blot method, it was found that the
nuclear probe was once again much closer to the actual situation, while the total
probe gives a marked underestimation.

The genes RTPi-66 and RTP2I-72 demonstrate the ability of the
• 25 nuclear probe to detect differentially expressed genes that do not appear
differentially with the total probe.

The genes for Nucleolin and Thrombospondin show that also for
down-regulated mRNAs the nuclear probe is much more sensitive and gives much
high levels of differential expression values.

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Lastly, the genes for ribosomal protein LI 7 and cytoplasmic

• 25

garama-actin are known as genes that do not respond to hypoxia stress. The nuclear
probe and the total probe both show that no induction occurs.

system for Polio virus 2A protease induced expression, since preliminary study
indicated that 2A protease enhances expression of IRES containing genes in this
cell line. HEK-293 cells were co-transfected with CMV-LacI - (constructed by
applicant using techniques known to those skilled in the art) in combination with
either one of the Polio virus 2 A protease expression vectors PTK-OP3-WT2A,
miniTK-WT2A, on PCIbb-LacI-Hyg (constructed by applicant on basis of vectors
from Stratagene) as shown in Figures 4A-C, respectively. The Lad expression
vector contained a hygromycin selectable marker, and the Polio virus 2A protease
expression vector contained a neomycin selectable marker which enabled the
isolation of clones resistant to both markers, presumably expressing both Lad
repressor and Polio virus 2A proteins.

Analysis of Polio virus 2 A protease expression

Death assay : - Resistant clones which grew after selection on hygromycin
(50^g/ml) and neomycin (500ng/ml), were treated with IPTG (5mM for 48h +
5mM for further 48h). Cells were then monitored for their viability and the clones
that showed full mortality upon Polio virus 2A protease induction, presumably
expressing the deleterious effect of the Polio virus 2A protease, were selected for
further analysis. Two such clones were isolated, HEK-293 cells expressing Polio
virus 2A protease under the control of a TK promoter (clone #14) and HEK-293
cells expressing the Polio virus 2A protease under the control of a miniTK
promoter (clone #1) as shown in Figure 5.

EXAMPLE 3

Identification of IRES Containing Genes

Establishment of mammalian cells expressing 2A protease

HEK-293 human (ATCC CRL-1573) cells were used as a model

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WO 99/58718 PCT7US99/10297
Analysis of 2 A protease expression : - Direct analysis of the Polio virus 2A
protease expression in HEK-293miniTK#l clones and HEK-293TK#14 clones
after IPTG induction was not performed due to the lack of antibodies against the
protein. Several currently available techniques can be used to measure changes in
gene expression including Northern blot analysis, RNase protection assay, in situ
hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). RT-
PCR is a very sensitive method, and was used to monitor the induction of the
mRNA encoding for Polio virus 2A protease in HEK-293miniTK# 1 clones
following IPTG treatment. mRNA was prepared from HEK-293 parental cells and
HEK-293 miniTK-2A clones following treatment with IPTG at different time
points. The RNAs were subjected to the RT-PCR reaction using Polio virus 2A
protease specific oligonucleotides:

5'GCAACTACCATTTGGCCACTCAGGAAG3', (SEQ ID No:28) and
5'GCAACCAACCCTTCTCCACCAGCAG3' and (SEQ ID No: 29).

Polio virus 2 A protease mRNA was not detected in HEK-293
parental cells, however it was induced following IPTG treatment and reached its
highest level after 48 hours of IPTG treatment as shown in Figure 6.
Analysis of 2 A protease activity

p220 cleavage : - A well characterized function of Polio virus 2 A protease is the
cleavage of the p220 protein (4Fy translational factor), a component of the 40S
ribosomal subunit. Cleavage of p220 yields three N-terminal cleavage products of
100-120KDa molecular weight due to post-translational modification. p220 and its
cleavage products were identified by 7% SDS PAGE and Western blot analysis
using polyclonal anti-p220 antibodies specifically directed against the N-terminal
region p220 as shown in Figure 6. Figure 6 demonstrates such an analysis in
which HEK-293 miniTK2A#l clone and HEK-293TK2A#14 clone were induced
for Polio virus 2 A protease expression to generate cleavage products of p220. As
control, HEK-293 cell lysate was treated with Polio virus 2 A protease produced by
in vitro translation, and was found to generate identical cleavage products with the
same mobility on 7% SDS PAGE as in the HEK-293 2A clones.

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WO 99/5871 8 PCT/US99/1 0297

This system was used as the source of mRNA for polysomal
fractionation. RDA analysis was performed using the protocol described above to
identify genes whose translation was up-regulated by the effects of the Polio virus
2A protease. Table 4 summarizes the results of analyses performed according to
5 the above-described method and genes isolated thereby.

Throughout this application various publications are referenced by
citation and patents by number. Full citations for the publication are listed below.
The disclosure of these publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the state of the art to
10 which this invention pertains.

The invention has been described in an illustrative manner, and it is
to be understood the terminology used is intended to be in the nature of description
rather than of limitation.

Obviously, many modifications and variations of the present
15 invention are possible in light of the above teachings. Therefore, it is to be

understood that within the scope of the appended claims, the invention may be
practiced otherwise than as specifically described.

TABLE 1

20 FRACTIONATION MEASURES AND IDENTIFIES

RNA associated with:

no fractionation changes of transcript abundance

Total RNA

Nuclear Measures denovo synthesis of mRNA

Cytoplasmatic Changes of transcript abundance

30 Cytoplasmatic/Nuclear transport of mRNA from the nucleus

Nuclear/Cytoplasmatic to the cytoplasm, increased or

decreased stability of mRNA

Polyribosomal/subpoly translationally controlled genes

35 ribosomal

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TABLE 1 - Continued

PCT/US99/10297

Rough Endoplasmic Reticulum
Microsomes

membrane bound polysomes
Cytoskeletal polyribosomes

mitochondrial

Splicesome

differences in the abundance of
transcripts encoding membrane and
secreted proteins

differences in abundance of transcript
encoding for nuclear proteins

differences in the abundance of
mRNA encoding mitochondrial
proteins

differences in alternative splicing

TABLE 2

Heat Shock Differential Gene Expression
Analysis with Polysomal Probes

clone

13h04

5b08

9fll

la04

13hl0

7c09

Hell

10c06

lb09

lc06

le09

3b04

13al2

7hl2

9dl2

13f09

9gl2

Ge " e Total

Pyruvate kinase No Change

Na,K-ATPase a-1 subunit No Change

^fP^ a No Change

FoIy(ADP-ribose) polymerase No Change

P™ 5 . Reduced x2

UWquitin Induced x2

Initiation Factor 4B No Change

90-kDa heat-shock protein No Change
Acyiamino acid-releasing enzyme No Change

P-spedrin Reduced x2

Elongation, £actor-l-gamxna No Change

Fibronectin Induced x2

Cytochrome C reductase core I No Change

Cytoskeletal y-actin No Change

Protein disulfide isomerase Reduced x2
DAPS

Polysome!
Induced »lo
Induced >10
Induced x4
Induced x4
Induced x5
Induced >6
Induced x4
Induced x4
Induced »10
Induced »10
Induced x5
Induced x4
Induced xlO
Induced >10
Induced >6
Induced >10
Induced xS

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WO 99/58718 PCT/US99/10297

TABLE 3

Statistics

Probe fJumber of differentials fold frdugfrm

Total mRNA 4hisHS 2 2

PolysomalRNA lhrHS 14 ^

8 "8

15 >M

10 37

Polysotnal RNA 4hrs HS 13. jJJ

18 >W
37

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WO 99/58718 PCI7US99/10297

TABLE 4

Translationally controlled genes
are identified by the 2A protease system

A. Rlbosomaf proteins or proteins directly Involved In
translation encoded by mRNAs containing 5' TOP#

S17 gbM13932
S9 gb U14971
EF-2 gbMl9997
L27a gb U14968
LS7a gbL06499

(Meyuhas et al. f 1996)

B. Proteins encoded by mRNAs containing 5TOP in their S'
UTR

Lamlnln binding receptor
p1-tubufln gb J00314

C. Gene with GC rich 5'UTR that regulates their
translation

spermidine synthase gbM34338
retlnol binding protein S'UTR X00129

D. Unknown genes potenlaly regulated by translation

E9T gbldS9051 EST gb AAQ431Q2 EST gbW76915

EST gbT54424 EST gb AAQ26896 0 45282

EST gbH16523 EST gb R07358

EST gbW96821 EST gb H83477

EST gbW99389 E9T T34436

E. Known genes that are potentially regulated by
translation (and may oonatfn IRES In their 5* UTR)

mitochondrial hinge protein gb661826
gp26L2 mitochondrial protein gp25L2
mHNA encoding a protein related to lyevl t-RNA
synthetase emb *31711 Y
SAP14 human spHceaqGome gb U41371

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PCT/US99/10297

REFERENCES CITED

Aiello et al., "Identification of multiple genes in bovine retinal pericytes altered by
exposure to elevated levels of glucose by using mRNA differential display" Proc.
Natl. Acad. Sci. USA Vol. 91. pp. 6231-6235 H994V

Bauer et al., "Identification of differentially expressed mRNA species by an
improved display technique (DDRT-PCR)" Nucleic Acids Research. Vol. 21, No.
18(1993).

Bharucha and Ven Murthy, "Characterization of Polysomes and Polysomal
mRNAs by Sucrose Density Gradient Centrifugation Followed by Immobilization
in Polyacrylamide Gel Matrix" Methods in Enzvmolo|gv. Vol. 216, pp. 168-179
(1992).

Braun et al., "Identification of Target Genes for the Ewing's Sarcoma EWS/FLI
Fusion Protein by Representational Difference Analysis" Molecular and Cellular
Biology, Vol. 15, No. 8, pp. 4623-4630 (1995).

Cold Springs Harbor Laboratory Press, Cold Spring Harbor, New York, 1989

Davis et al., " Expression of a single transfected cDNA converts fibroblasts to
myoblasts." Cell 51:987-1000, 1987.

Diatchenko et al., "Suppression subtractive hybridization: A method for
generating differentially regulated or tissue-specific cDNA probes and libraries"
Proc. Natl Acad. Sci., Vol. 93, pp. 6025-6030 (1996).

Ehrenfeld, "Initiation of Translation by Picornavirus RNAs", Translational Control
Cold Spring Harbor Laboratory Press, pp. 549-573, 1996.

Hadman et al., "Modification to the differential display technique reduce
background and increase sensitivity" Analytical Biochemistry 226:383-386 (1995).

Hanauske-Abel et al., "Detection of a sub-set of polysomal mRNAs associated
with modulation of hypusine formation at the Gl-S boundary. Proposal of a role
for EIF-5A in onset of DNA replication." FEBS Letters 386 pp. 92-98 (1995).

Hirama et al., "Direct Purification of Polyadenylated RNAs from Isolated
Polysome Fractions" Analytical Biochemistry. 155, pp. 385-390 (1986).

Hubank and Schatz, "Identifying differences in mRNA expression by
representational difference analysis of cDNA" Nucleic Acids Research. V ol. 22,
No. 25, p. 5640-5648 (1994).

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WO 99/587 1 8 PCT/US99/1 0297

Jefferies et al., "Elongation Faction-la mRNA Is Selectively Translated following
Mitogenic Stimulation" The Journal of Biological Chemistry. Vol. 269, No. 6, pp.
4367-4372 (1994).

5 Liang and Pardee, "Differential Display of Eukaryotic Messenger RNA by Means
of the Polymerase Chain Reaction" Science, Vol. 257, pp. 967-971 (1992).

Liang et al., "Distribution and cloning of Eukaryotic mRNAs by means of
differential display: refinements and optimization" Nucleic Acids Research Vol.
10 21, No. 14, pp. 3269-3275 (1993).

Liang and Pardee, "Recent advances in differential display" Current Opinion in
Immunology, 7:274-280 (1995V

15 Linskens et al., Cataloging altered gene expression in young and senescent cells
using enhanced differential display" Nuc. Ac. Res .
23:3244-3251 (1995).

Lisitsyn and Wigler, "Cloning the Differences Between Two Complex Genomes"
20 Science, Vol. 259, pp. 946-951 (1993).

Mach et al., "Isolation of a cDNA Clone Encoding S-Adenosylmethionine
Decarboxylase" The Annual of Biological Chemistry, Vol. 261, No. 25, pp. 1 1697-
11703(1986).

Macejak et al., "Internal inition of translation mediated by the 5' leader of a cellular
mRNA" Nature, Vol. 353, pp. 990-94 (1991).

Mechler, "Isolation of messenger RNA from Membrane-Bound Polysomes"
30 Methods in Enzvmology. Vol. 152, pp. 241-253 (1987).

Menaker et al., "A Method for the Isolation of Rat Submandibular Salivary Gland
Polysomes on Linear Sucrose Density Gradients" Analytical Biochemistry 57, pp.
325-335 (1974).

Meyuhas et al., "Translational Control of Ribosomal Protein mRNAs in
Eukaryotes" Translational Control, pp. 363-388 (1996).

Mountford et al., "Internal ribosome entry sites and dicistronic RNAs in
40 mammalian transgenesis" TIG, Vol. 1 1. No. 5, pp. 179-184 (1995).

Ogishima et al., "Fractionation of Mammalian Tissue mRNAs by High-
Performance Gel Filtration Chromatography" Analytical Biochemistry. 138, pp.
309-313 (1984).

45 Oh et al., "Gene regulation: translational initiation by internal ribosome binding"
Current Opinion in Genetics and development pp. 295-300 (1993).

.34-

WO 99/58718

PCT/US99/10297

Pelletier et al., "Internal initiation of translation of eukaryotic mRNA directed by a
sequence derived from poliovirus RNA" Nature, Vol. 334, pp. 320-325 (1988).

Schena et al., "Quantitative Monitoring of Gene Expression Patterns with a
Complementary DNA Microarray" Science, Vol. 270, pp. 467-470 1995).

Shen et al., "Identification of the Human Prostatic Carcinoma Oncogene PTI-1 by
Rapid Expression Cloning and Differential RNA Display" Proc. Natl. Acad. Sci.
USA, Vol. 92, pp. 6778-6782 (1995).

Vagner et al. "Alternative Translation of Human Fibroblast Growth Factor 2
mRNA Occurs by Internal Entry of Ribosomes" Molecular and Cellular B iology.
Vol. 15,No. 1, pp. 35^4(1995).

Welsh et al., "Arbitrary primed PCR fingerprinting of RNA",
Nuc. Ac. Res . 20:4965-4970 (1992).

Zhao et al., "New primer strategy improves precision of differential display"
Biotechniaues 18: 842-850 (1995).

-35-

WO 99/5871 8 PCT/US99/1 0297

CLAIMS

What is claimed is:

1 . A method or process for identifying genes whose expression is
5 responsive to a specific cue or cues including the steps of:

(a) applying a cue to an organism or tissue or cells;

(b) isolating specific cellular fractions from the tissues or cells
subjected to the cue;

10 (d) differentially analyzing the mRNA samples in comparison with

control samples not subjected to the cue to identify genes that have responded to
the cue.

2. A method as set forth in claim 1, wherein the cue is a toxin or a
15 chemical, or a pharmaceutical, or a mechanical stress, or an electric current, or a

pathogen or a pathological condition, or a hormone, or a specific protein.

3 . The method as set forth in claim 2, wherein said cue is further
defined as chemically treating the cells, or irradiating the cells, or depriving the

20 cells of oxygen.

4. A method as set forth in claim 2, wherein the cue is further
defined as a stress-inducing element of unknown relationship to gene translation.

25 5. A method as set forth in claim 1 , wherein genes are identified at

the translation level; genes regulated at the transcription level; genes regulated by
RNA stability; genes regulated by mRNA transport rate between the nucleus and
cytoplasm; genes regulated by differential splicing; and genes regulated by
antisense RNA.

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WO 99/58718 PCT/US99/1 0297

6. A method as set forth in claim 1, wherein the mRNA samples are
further fractionated into mRNA subfractions which are subjected to differential
analysis to identify genes responsive to the cue at all levels of expression
regulation as herein defined and to determine the abundance and direction of the
response.

7. A method as set forth in claim 6, wherein the mRNA sample is
fractionated into one or more subfractions from the group consisting essentially of
cytoplasmic, nuclear, polyribosomal, sub polyribosomal, microsomal or rough
endoplasmic reticulum, mitochondrial and splicesome associated mRNA.

8. A method as set forth in claim 1, wherein said differential
analysis step is selected from the group consisting of differential display,
representational differential analysis (RDA), suppressive subtraction hybridization
(SSH), serial analysis of gene expression (SAGE), gene expression microanay
(GEM), nucleic acid chip technology, oligonucleotide chip technology; DNA
membrane arrays; direct sequencing and variations and combinations of these
methods.

9. A method as set forth in claim 8, wherein said differential
analysis step is further defined as identifying and measuring the genes regulated at
the translation level.

10. A method as set forth in claim 8, wherein said differential
analysis step is further defined as identifying and measuring the genes regulated at
the transcription level.

1 1 . A method as set forth in claim 8, wherein said differential
analysis step is further defined as identifying and measuring the genes regulated by
RNA stability.

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WO 99/58718 PCT/US99/10297
12. A method as set forth in claim 8, wherein said differential
analysis step is further defined as identifying and measuring the genes regulated by
mRNA transport rate between the nucleus and the cytoplasm.

5 13. A method as set forth in claim 8, wherein said differential

analysis step is further defined as identifying and measuring the genes regulated by
differential splicing.

14. A method as set forth in claim 8, wherein said differential

10 analysis step is further defined as identifying and measuring the genes encoding
secreted and membrane proteins.

15. A method as set forth in claim 8, wherein said differential
analysis step is further defined as identifying and measuring the genes encoding for

1 5 nuclear proteins.

16. A method for identifying gene sequences coding for internal
ribosome entry sites, said method comprising the steps of:

inhibiting 5 'cap-dependant mRNA translation in a cell;
20 collecting a pool of mRNA from the cells; and

differentially analyzing the pool of mRNA to identify genes with
sequences coding for internal ribosome entry sites.

17. A method as set forth in claim 16, wherein said inhibiting step
25 is further defined as selecting for non-5'-cap dependent mRNA translation.

18. A method as set forth in claim 16, wherein said inhibiting step
further includes the step of incorporating a gene coding for Polio virus 2A protease
into the cell.

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WO 99/58718

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-25

1 9. A method as set forth in claim 1 8, wherein said incorporation
step is further defined as transforming the cell with a vector containing the gene
coding for the Polio virus 2A protease.

20. A method as set forth in claim 1 8 including the step of
controlling the expression of the gene coding for the Polio virus 2A protease.

21. A method as set forth in claim 16, wherein said analyzing step
is further defined as differential display analysis.

22. A method as set forth in claim 16, wherein said analyzing step
is further defined as representational difference analysis.

23. A method as set forth in claim 16, wherein said analyzing step
is further defined as performing a gene expression microarray analysis.

24. A method as set forth in claim 16, including the further step of
cloning genes identified as being translationally regulated.

25. A method as set forth in claim 16, wherein said analyzing step
distinguishes between polysomal fractions that migrate in the same density
individually or in a pool.

26. A method as set forth in claim 16, wherein said analyzing step
distinguishes between nonpolysomal fractions individually or as a pool.

distinguishes between stimulated polysomal and nonpolysomal fractions
individually or in a pool.

27. A method as set forth in claim 16, wherein said analyzing step

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WO 99/58718 PCT/US99/10297
28. A method as set forth in claim 16, wherein said analyzing step
distinguishes between each of the polysomal and nonpolysomal fractions
individually or in a pool compared to an unfractionated total RNA pool.

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non-polysomal
o o o

mRNP><o*> polyribosomes

►

Sedimentation

Fig-IA

Fig- IB

SUBSTITUTE SHEET (RULE 26)

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4hr> ifihrs
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Tot HQ 4.? QE 7 .6

stp BE! 14.2 BH.-'.i

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4hrs 16hrs
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Tot 3.4 HLJ 3.4
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4hrs 16hrs
X H X H

Tot i " sao -

STP BE3 2.5 EfiJ "

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4hr> 16hrs
X H X H
Tot BE? 2.6 E3E 4.8
STP OB -2.8 BE -3 2

15F0?
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4hrs I6hrs
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Tot BIS i.i MB*

STP BB3.4 BB3.0

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stp BB -1.2 &B i.s

ng-3

SUBSTITUTE SHEET (RULE 26)

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4/7

Fspl 5223
Bgll 5121

Pvull 800

Fspl 804
Pstl 857

Pstl 1054
EcoRI 1143

Pvull 3932

Xhol 3575
C/oi 3561
(Sail) 3569
(Hindlll) 3554

1849

Fspl 1856
Pvull 1906

'Xbai 3298i
Psll 3138
(BamHI) 3092]
Spel 3086i
(Xbal) 3080
Moil 3073
(Hindlll) 3066
(BamHI) 3031

Hindlll 2585 s
Clal 2590
(Hindlll) 2599
(Sail) 2691
(Sail) 2734
tSall) 2777
(BamHI) 2808

-Xbal 2065
-BamHI 2075

Fig-4A

Fspl 4959
Bgll 4857-

Pvull 800
Fspl 804
Pstl 857

Psil 1054
EcoRI

Pvull 3668"
Xhol 331 1

Clal 3297-
(Sall) 3305'

(Hindlll) 3290

H43

Xbal 3034
Psil 2874

Bgll 1849
'Fspl 1856
Pvull 1906
Sad 2053

BamHI 2062
Xbal 2073
Hindlll 2107
, ,-™JU><>/ 2134
Xhol 2300^ BamHI 2139
(Hindlll) 2335
(Sail) 2427

(Sail) 2470

(Sail) 2513
(BamHI) 2544
(BamHI) 2767
(Hindlll) 2802
Noll 2809

Fig-4B

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Pvul(2) 6017
Scal(2) 5905
Xmnl 5786

Dralll 4938
Noel 4832
NgoMI 4830
Pvul(2) 4638
Ascl(2) 4619
BamHI 4612
Clal 4605
Hindlll 4603

Asci(2) 7293
Bglll 7286 ?P e '

Sad 721

Hindlll 748
Pstl 830

Aflll(2) 820
1017

Scal(2) 1030
Nhel 1052

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Mlul 1069
#pnl 1079
Xbal 1080

Fig-4C

0 24 48 72 96 120

1IUZ AFTER 2A INDUCTION (HOURS)

Rg-5

SUBSTITUTE SHEET (RULE 26)

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Induction of

2 A transcription

s 1 I 293-2A

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. 0) W

SUBSTITUTE SHEET (RULE 26)

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT; Luria, Sylvie

Einat, Paz
Harris, Nicholas
Skaliter, Rami
Grosman, Zehava

(ii) TITLE OF INVENTION: METHOD FOR IDENTIFYING TRANS LATIONALLY
REGULATED GENES

(iii) NUMBER OF SEQUENCES : 29

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Kohrt & Associates

(B) STREET: 30500 Northwestern Hwy. , Suite 410

(D) STATE: Michigan

(E) COUNTRY: US

(F) ZIP: 48334

(v) COMPUTER READABLE FORM:

(A) ME DIUM T YPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/ AGENT INFORMATION:

(A) NAME: Kohn, Kenneth I.

(B) REGISTRATION NUMBER: 30,955

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (248) 539-5050

(B) TELEFAX: (248) 539-5055

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CATTATGCTG AGTGATATCT 7TTTTTTTVV

WO 99/58718 PCT/US99/10297

CJ INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE : other nucleic acid
(A) DESCRIPTION: /desc = "Primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CATTATGCTG AGTGATATCT TTTTTTTTAA

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CATTATGCTG AGTGATATCT TTTTTTTTAC

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : 1 inear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 4 :

CATTATGCTG AGTGATATCT TTTTTTTTAG

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii)' MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(Xi). SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

CATTATGCTG AGTGATATCT TTTTTTTTCA

WO 99/5871 8 PCI7US99/1 0297

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENC 2 CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CATTATGCTG AGTGATATCT TTTTTTTTCC

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CATTATGCTG AGTGATATCT TTTTTTTTCG

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc » "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :

CATTATGCTG AGTGATATCT TTTTTTTTGA

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B x TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc « "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:.

CATTATGCTG AGTGATATCT TTTTTTTTGC

WO 99/58718 PCT/US99/10297

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

Ui) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CATTATGCTG AGTGATATCT TTTTTTTTGG

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

ATTAACCCTC ACTAAANNNN NNNNNN

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "primer

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
ATTAACCCTC ACTAAATGCT GGGGA

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE : other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(Xi) SEQUENCE DESCRIPTION: S£Q ID NO: 13

ATTAACCCTC ACTAAATGCT GGAGG

WO 99/58718 PCT/US99/10297

(2) INFORMATION FOR SEQ ID NC:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc « "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

ATTAACCCTC ACTAAATGCT GGTAG

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc * "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

ATTAACCCTC ACTAAATGCT GGTAG

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

ATTAACCCTC ACTAAAGATC TGACTG

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

ATTAACCCTC ACTAAATGCT GGGTG

WO 99/58718 PCT/US99/1 0297

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ATTAACCCTC ACTAAATGCT GTATG

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

ATTAACCCTC ACTAAATGGA GCTGG

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc * "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: .20:

ATTAACCCTC ACTAAATGTG GCAGG

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi). SEQUENCE DESCRIPTION: SEQ ID NO:2I:

ATTAACCCTC ACTAAATGCA CCGTCC

PCI7US99/10297

{2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid
.[•?) STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

GATCTGCGGT GA

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid
<C) STRANDEDNESS : single
<D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc « "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

AGCACTCTCC AGCCTCTCAC CGCA

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GATCTGTTCA TG.

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc * "primer"

(xi: SEQUENCE DESCRIPTION: SEQ ID NO: 25:

ACCGACGTCG ACTATCCAT3 AACA

PCT/US99/10297

(2) INFORMATION FOr. SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

GATCTTCCCT CG

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc - "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

AGGCAACTGT GCTATCCGAG GGAA

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = -primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

GCAACTACCA TTTGGCCACT CAGGAAG

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION : SEQ ID NO: 29:

GCXACCAAC- CTTCTCCACC AGCAG

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US99/I0297

A. CLASSIFICATION OF SUBJECT MATTER

IPC(6) :C12Q 1/68; C12P 19/34; C07H 21/04; C07K 13/00

US CL : 435/6, 91.2. 91.51; 536/243; 530/350
According to International Patent Classification QPC) or to both national classification and IPC

a FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

U.S. : 435/6. 91.2. 91.51; 536/24.3; 530/350

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
Please See Extra Sheet

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US 5,525,471 A (ZENG) 11 JUNE 1996, the Abstract, col. 5, lines
5-20 and 29-49, col. 10, lines 31-53.

US 5,459,037 A (SUTCLIFFE et al.) 17 OCTOBER 1995, the
Abstract, col. 16, lines 28-39, col. 19, lines 29-35, col. 20, lines 1-
34.

1-15
1-15

[ | Further documents are listed in the continuation of Box C. ["*"] See patent family annex.

■ of cited dot

later do**

document dsfinmf the general state of the art which » not considered
to be of pvtooukr relevance

car her document publrshcd oa or sfUr the mtcrnstioneJ films date

document which may throw doubts oa priority ehnm(s) or which ia
ciud to astabtkh the publication data of another citation or other
eon (as specified)

raf erring to an a

document published prior to the

filing date but later than

but cited to i

Dent of particular relevance; the churned nircut io n e
considered novel or esmics be eonaidared to involve an invci
when the document ia taken alone

document of particular relevance; the churned mvenhon
considered to involve en inventive step when tbm '
combined with one or more other such docu
borne; obvious to e person skilled in the art

document member of the same patent family

Date of the actual completion of the international search
29 JULY 1999

Date of mailing of the international search report

1 8 AUG 1999

Name and mailing address of the ISAAJS
Commissioner of Patents and Trademark*
Box PCT

Washington, D.C 20231
Facsimile No. (703) 305-3230

Authorize^fTiccjj/ y

JOCYE TUNG ytPL-
Telephone No. (703) 308-0 196

Form PCT/ISA/210 (second sbeetXJuly 1992)*

INTERNATIONA L SEARCH REPORT

International application No.
PCT/US99/10297

Box I Observations where certain claims were found unsesrchable (Continuation of item 1 of first sbeet)

This international report has not been established in respect of certain claims under Article 17(2X*) for the following reasons:
1. |~| Claims Nos.:

1—1 because they relate to subject matter not required to be searched by this Authority, namely:

2. [""] Claims Nos.:

' — ' because they relate to parts of the international application that do not comply with the prescribed requirements to such
an extent that no meaningful international search can be carried out, specifically:

3. Claims Nos.:

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:
Please See Extra Sheet

1. PI As all required additional search fees were timely paid by the applicant, this international search report covers all searchable
L "" 1 claims.

2. ^] As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment

of any additional fee.

3. [ | As only some of the required additional search fees were timely paid by the applicant, this international search report covers

only those claims for which fees were paid, specifically claims Nos.:

4. No required additional search fees were timely paid by the applicant Consequently, this international search report is

restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest The additional search fees were accompanied by the applicant's protest

[~| No protest accompanied the payment of additional search fees.

Form PCT/lSA/210 (continuation of first sheet(l)XJuty 1992)*

INTERNATIONAL SEARCH REPORT

International application No.

PCT/US99/10297

B, FIELDS SEARCHED

Electronic data bases consulted (Name of data base and where practicable terms used):
uspat, medline, biosis, caplus

search terms: identify genes, gene expression, differential analysis, mRNA, translation, transcription, RNA stability,
splicing, antisense RNA, representational differential analysis, suppressive subtraction hybridization, sequencing, nucleic
acid chip

BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING
This ISA found multiple inventions as follows:

This application contains the following inventions or groups of inventions which are not so linked as to form a single
inventive concept under PCT Rule 13.1. In order for all inventions to be searched, the appropriate additional search fees
must bo paid.

Group I, claim(s)MS, drawn to a method or process for identifying genes whose expression is responsive to a specific
cue.

Group II, claim(s) 16-28, drawn to a method for identifying gene sequence coding for internal ribosome entry sites.

The inventions listed as Groups I and II do not relate to a single inventive concept under PCT Rule 13.1 because, under
PCT Rule 13.2, they lack the same or corresponding special technical features for the following reasons: Group I is
drawn to a method for identifying genes whose expression is responsive to a specific cue involving identifying the genes
at the translation level and transcription level (see details in claim 5) and the differential analysis step is representational
differentia] analysis and suppressive subtraction hybridization (see details in claim 8), while Group II is drawn to identify
gene sequence coding for internal ribosome entry sites in which the method steps are different from the method steps of
Group 1, for example, inhibiting 5* cap-dependant mRNA translation in a cell. Thus, the instant invention lacks the same
special technical features.

Form PCT/ISA/210 (extra sheetXJuly 1992)*

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